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HEK293 表达的Nanodisc / DDM全长CD20蛋白

FACS Verified and Protocols Offered

背景

Lionel Rougé, et al. Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab. Science. 2020
2020年2月20日Genentech在Science上发表的一篇文章Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab,突破性地解析了全长CD20蛋白以及CD20蛋白与Rituximab Fabs复合物的三维结构。文章证实Rituximab对CD20的识别不止有一级表位ECL2,还识别其二级表位ECL1/ECL2。SPR验证的Rituximab与全长CD20蛋白以及与ECL2多肽表位的亲和力差异,表明二级表位ECL1/ECL2会影响Rituximab和CD20的亲和力。另外文章发现二级表位ECL1/ECL2还会影响Rituximab的CDC效应,揭示了具有完整结构的全长CD20蛋白的重要意义。(页面尾部可下载文献全文)
在2019年召开的第61届美国血液学会(ASH)报道了CD20-CD3 T细胞双特异性抗体CD20-TCB(RG6026)同时靶向B细胞表面的CD20和T细胞表面的CD3,其治疗R/R B-NHL的2项临床研究初步结果显示该双特异性抗体具有可接受的安全性以及有前景的疗效。
CD20是淋巴癌、白血病和某些自身免疫等疾病治疗的热门目标靶点,其在免疫治疗领域的应用非常广泛。但因为全长CD20蛋白的表达难度高、活性不稳定等因素,目前市面上很难找到合适的CD20蛋白用于活性分析。
为了解决这些问题,ACROBiosystems采用独特的设计开发了全球独有的由HEK293表达的多次跨膜全长CD20蛋白,包括未进行标记的和生物素标记的Nanodisc或DDM不同版本产品。全长CD20蛋白同时有small loop和big loop,具有完整的天然正确构象,可用于免疫/ ELISA/ SPR/ BLI/ FACS。我们验证了Rituximab、Ofatumumab与全长CD20蛋白的结合活性,全长CD20蛋白展现出纳摩尔级别的高亲和力,可免费提供Protocol。
不同类型的全长CD20蛋白
DDM版本
DDM是一种非离子型去垢剂,能够使脂膜解体释放膜蛋白,并在溶液中为去膜状态下的膜蛋白提供疏水环境,维持和保护膜蛋白的疏水跨膜结构。但DDM的存在会破坏细胞膜的结构,一般不用推荐于细胞实验。
Nanodisc版本(不含去垢剂)
Nanodisc (纳米磷脂盘) 是由膜支架蛋白(membrane scaffold proteins, MSPs)和磷脂分子构成的磷脂双分子层类膜结构,膜蛋白可以整合到Nanodisc的特殊结构中,保持其生物学活性并有很好的水溶性,应用更广泛。ACROBiosystems使用的Nanodisc技术已经获得专利持有方的授权,生物药研发企业在研发过程中可放心使用。

Ilia G. Denisov, et al., Nanodisc in membrane biochemistry and biophysics. Chem Rev. 2017(页面尾部可下载文献全文)

产品列表
ACRO开发的生物素标记的全长CD20蛋白(DDM和Nanodisc)经FACS验证具有高生物活性,点击下方可申请protocol。
Molecule Cat. No. Host Type Product Description
CD20/ MS4A1 CD0-H52H1 HEK293 Unconjugated Human CD20 / MS4A1 Full Length Protein, His Tag (Nanodisc) (HEK293)
Request FACS Protocol
CD0-H52H3 HEK293 Unconjugated Human CD20 / MS4A1 Full Length Protein, His Tag (HEK293) (SPR verified)
Request FACS Protocol
CD0-H82E3 HEK293 Biotin-labeled Biotinylated Human CD20 / MS4A1 Full Length Protein, His,Avitag™ (Nanodisc) (HEK293)
Request FACS Protocol
CD0-H82E5 HEK293 Biotin-labeled Biotinylated Human CD20 / MS4A1 Full Length Protein, His,Avitag™ (HEK293)
Request FACS Protocol
产品特点
全长CD20蛋白由真核系统表达具有完整的天然构象(同时具有small loop和big loop)
全长CD20蛋白能提供更好的活性

A) HEK293 Cell (Full Length)

Immobilized Rituximab at 2 μg/mL (100 μL/well) can bind Human CD20 Full Length, His Tag, HEK293 (SPR verified) (Cat. No. CD0-H52H3) with a linear range of 0.4-3 ng/mL (in presence of DDM and CHS).

Protocol

B) E.coli (Extracellular)

Immobilized Rituximab at 5 μg/mL (100 μL/well) can bind Human CD20, TrxA Tag, His Tag (E.coli) with a linear range of 78-2500 ng/mL.

ACRO的全长CD20蛋白活性经FACS/ELISA/ SPR验证,可免费提供相应的Protocol
FACS验证
Cat. No. CD0-H82E3 (HEK293表达)
FACS Analysis of anti-CD20 CAR expression

2e5 of CD20-CAR-293 cells transfected with anti-CD20-scFv were stained with 100 μL of 3 μg/mL of Biotinylated Human CD20 Full Length, His,Avitag (Cat. No. CD0-H82E3) and negative control antibody respectively, washed and then followed by PE-SA and analyzed with FACS.

Protocol

Cat. No. CD0-H52H1 (HEK293表达)
FACS Analysis of anti-CD20 CAR expression

2e5 of CD20-CAR-293 cells transfected with anti-CD20-scFv were stained with 100 μL of 3 μg/mL of Human CD20 Full Length, His Tag (Cat. No. CD0-H52H1) and negative control antibody respectively, washed and then followed by PE anti-His antibody and analyzed with FACS.

Protocol

ELISA验证
Cat. No. CD0-H82E3 (HEK293表达)

Immobilized Rituximab at 5 μg/mL (100 μL/well) can bind Biotinylated Human CD20 Full Length, His,Avitag (Cat. No. CD0-H82E3) with a linear range of 0.008-0.125 μg/mL

Protocol

Cat. No. CD0-H52H1 (HEK293表达)

Immobilized Rituximab at 2 μg/mL (100 μL/well) can bind Human CD20 Full Length, His Tag (Cat. No. CD0-H52H1) with a linear range of 1-63 ng/mL

Protocol

Cat. No. CD0-H82E5 (HEK293表达)

Immobilized Rituximab at 2 μg/mL (100 μL/well) can bind Biotinylated Human CD20 Full Length, His,Avitag (HEK293) (Cat. No. CD0-H82E5) with a linear range of 4-63 ng/mL (in presence of DDM and CHS).

Protocol

Cat. No. CD0-H82E5 (HEK293表达)

Immobilized Ofatumumab at 2 μg/mL (100 μL/well) can bind Biotinylated Human CD20 Full Length, His,Avitag (HEK293) (Cat. No. CD0-H82E5) with a linear range of 4-63 ng/mL (in presence of DDM and CHS).

Protocol

Cat. No. CD0-H52H3 (HEK293表达)

Immobilized Rituximab at 2 μg/mL (100 μL/well) can bind Human CD20 Full Length, His Tag, HEK293 (SPR verified) (Cat. No. CD0-H52H3) with a linear range of 0.4-3 ng/mL (in presence of DDM and CHS).

Protocol

Cat. No. CD0-H52H3 (HEK293表达)

Immobilized Ofatumumab at 2 μg/mL (100 μL/well) can bind Human CD20 Full Length, His Tag, HEK293 (SPR verified) (Cat. No. CD0-H52H3) with a linear range of 0.4-6 ng/mL (in presence of DDM and CHS).

Protocol

SPR验证
Cat. No. CD0-H82E5 (HEK293表达)

Biotinylated Human CD20, His,Avitag (HEK293) (Cat. No. CD0-H82E5) captured on Biotin CAP-Series S Sensor Chip can bind Rituximab with an affinity constant of 1.73 nM as determined in a SPR assay (in presence of DDM and CHS) (Biacore T200).

Protocol

Cat. No. CD0-H52H3 (HEK293表达)

Human CD20 Full Length, His Tag, HEK293 (SPR verified) (Cat. No. CD0-H52H3) captured on CM5 chip via Anti-human IgG Fc antibodies surface can bind MabThera® (Rituximab) with an affinity constant of 6.21 nM as determined in a SPR assay (in presence of DDM and CHS) (Biacore 8K).

Protocol

产品评论
 
评论(3)
  1. 158XXXXXXX9
  2. 10人赞
  3. BLI实验。相比之前在另一家公司买的CD20蛋白,这个蛋白结合活性更强,CD20这种特殊膜蛋白,能够全长表达还具有生物活性,还是可以的。就是价格能稍微便宜点就更好了
  4. 2019-10-23
  1. 150XXXXXXX0
  2. 9人赞
  3. CD20因为是多次跨膜蛋白,找了很多家公司都表达不好。 抗体筛选和Elisa都定了很多次,实验效果不错。 除了有点小贵没有别的缺点啦!
  4. 2019-10-11
  1. 177XXXXXXX3
  2. 12人赞
  3. 重组抗CD20单克隆抗体的靶点受体是跨膜蛋白,且体外表达的CD20受体很难维持其生物活性,我们常采用细胞法或流式方法进行活性评价,其涉及细胞制备,周期比较长,重复性不是很好。因此我们迫切需要建立SPR及ELISA分析方法对重组抗CD20单克隆抗体结合活性进行评价,而这个体外表达的CD20受体蛋白正好满足我们需求,并且已对其实用性进行验证。对加快我门产品研发进度,非常有帮助!
  4. 2019-10-11
 
相关参考文献

Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab

Nanodisc in membrane biochemistry and biophysics.

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